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Department of Pharmacology and Toxicology, College of Pharmacy, the University of Arizona, Tucson, Arizona
Received February 4, 2005; accepted April 18, 2005
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Abstract |
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). After the initial cloning of these receptors, their functional significance was unclear because they both were preferentially activated by PGE2 and they both seemed to signal exclusively through the activation of adenylyl cyclase (Honda et al., 1993; Regan et al., 1994). It was subsequently determined, however, that they differed with respect to PGE2-mediated receptor desensitization and internalization. Thus, EP4 receptors were found to undergo rapid agonist-induced desensitization, whereas EP2 receptors did not (Nishigaki et al., 1996). Likewise, EP4 receptors were found to undergo rapid PGE2-mediated internalization, and EP2 receptors did not (Desai et al., 2000).
The first evidence of differences in the signaling potential of the EP2 and EP4 receptors involved the observation that PGE2 could stimulate the phosphorylation of glycogen synthase kinase-3 (GSK-3) and T cell factor (Tcf) transcriptional activation in cells stably expressing these receptors (Fujino et al., 2002). Although both receptors possessed these activities, the stimulation of GSK-3 phosphorylation and Tcf transcriptional activation by the EP2 receptor was primarily through a PKA-dependent pathway, whereas, for the EP4 receptor, these effects were mediated primarily through a phosphatidylinositol 3-kinase (PI3K)-dependent pathway. It was further shown that PGE2 treatment of EP4-expressing cells, but not EP2-expressing cells, resulted in the induction of early growth response factor-1 (EGR-1) by a pathway involving the activation of PI3K and the extracellular signal-regulated kinases (ERKs) (Fujino et al., 2003). Additional evidence of EP4 receptor signaling through a PI3K-dependent pathway has also been reported for colorectal carcinoma cells. Thus, PGE2 was found to increase the growth and motility of human adenocarcinoma cells (LS-174) through activation of PI3K via stimulation of EP4 receptors (Sheng et al., 2001). Likewise, it has been reported that stimulation of EP4 receptors by PGE2 in mouse colon adenocarcinoma cells (CT26) activates PI3K and ERKs signaling and is associated with cell growth in the absence of any detectable increase in intracellular cAMP formation (Pozzi et al., 2004).
An important function of G
s-coupled receptors is the transcriptional regulation of genes whose promoters contain cAMP response elements (CREs). In this signaling cascade, the release of Gs after stimulation of the receptor leads to the activation of adenylyl cyclase and increased formation of intracellular cAMP. The subsequent activation of PKA by cAMP can result in the phosphorylation of the CRE binding protein (CREB), which is a transcription factor that interacts with CREs and is central to the regulation cAMP responsive gene expression (Mayr and Montminy, 2001; Johannessen et al., 2004). Among the many genes whose expression can be regulated by cAMP is cyclooxygenase-2, whose catalytic product, PGH2, is the immediate precursor for the biosynthesis of the prostaglandins and thromboxanes. It is interesting that bradykinin has been found to increase cAMP-dependent cyclooxygenase-2 promoter activity in human pulmonary artery smooth muscle cells through an autocrine signaling pathway involving the activation of endogenous EP2 and EP4 prostanoid receptors (Bradbury et al., 2003).
The phosphorylation of CREB by PKA occurs at serine-133 and results in the recruitment of the CREB-binding protein and/or its paralogue, p300, which function with phospho-CREB as coactivators of gene transcription (Mayr and Montminy, 2001; Johannessen et al., 2004). However, phosphorylation at Ser-133 does not occur exclusively by way of cAMP signaling and PKA. For example, CREB may be phosphorylated at Ser-133 by the calcium/calmodulin-dependent kinases and by members of the pp90rsk family kinases after the activation of calcium signaling and growth factor-mediated mitogenic signaling, respectively (Mayr and Montminy, 2001; Johannessen et al., 2004). In addition, the phosphorylation of CREB at Ser-133 has been reported to occur in a PI3K-dependent manner after the activation of the ERKs and Akt signaling pathways (Mayr and Montminy, 2001; Johannessen et al., 2004).
Given the ability of EP2 and EP4 receptors to activate cAMP signaling pathways and the additional ability of EP4 receptors to activate PI3K signaling pathways, we were interested in the potential phosphorylation of CREB at Ser-133 by these receptors. We now show that stimulation of both the human EP2 and EP4 receptors by PGE2 can lead to the phosphorylation of CREB at Ser-133. In EP2-expressing cells, the mechanism is primarily cAMP- and PKA-dependent. In EP4-expressing cells, the mechanism is more complex and involves a PI3K-dependent pathway. A novel finding is that PGE2 stimulation of EP4-expressing cells negatively regulates the activity of PKA through the activation of PI3K signaling.
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Materials and Methods |
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, 2003). Cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 250 µg/ml G-418 (Geneticin), 100 µg/ml gentamicin, and 200 µg/ml hygromycin B (all from Invitrogen). Sixteen hours before the immunoblotting experiments, cells were switched from their regular Dulbecco's modified Eagle's medium to Opti-MEM (Invitrogen) containing 250 µg/ml G-418 and 100 µg/ml gentamicin. Cells were incubated at 37°C with either vehicle (0.1% Me2SO) or 1 µM PGE2 (Cayman) for the time indicated in the figures. For experiments involving the use of signaling inhibitors, cells were pretreated with either vehicle (0.1% Me2SO) or with 10 µM H-89 (Calbiochem) or with 100 nM wortmannin (Sigma) or with the combination of 10 µM H-89 and 100 nM wortmannin for 15 min at 37°C. Cells were then scraped into a lysis buffer consisting of 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, pH 8.0, 1% Nonidet P-40, 0.5% sodium deoxycholate, 10 mM sodium fluoride, 10 mM disodium pyrophosphate, 0.1% SDS, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 10 µM leupeptin, and 10 µg/ml aprotinin and transferred to microcentrifuge tubes. The samples were rotated for 30 min at 4°C and were centrifuged at 16,000g for 15 min. Aliquots of the supernatants containing 20 µg of protein were electrophoresed on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes as described previously (Fujino et al., 2002; Fujino et al., 2003). Membranes were incubated in 5% nonfat dry milk for 1 h and were then washed and incubated for 16 h at 4°C in 5% bovine serum albumin containing anti-phospho-CREB (Ser-133) antibody (Cell Signaling Technology, Beverly, MA) at a dilution of 1:1000. Membranes were washed three times and incubated for 1 h at room temperature in 5% nonfat milk with a 1:2000 dilution of anti-rabbit secondary antibodies conjugated with horseradish peroxidase. After three washes, immunoreactivity was detected by chemiluminescence as described previously (Fujino et al., 2002, 2003). To ensure equal loading of proteins, the membranes were stripped and reprobed with a 1:1000 dilution of anti-CREB antibody (Cell Signaling Technology) as described above for the anti-phospho-CREB antibody.
PKA Kinase Activity Assay. Cells were cultured in 12-well plates and were pretreated with either vehicle (0.1% Me2SO) or inhibitors (10 µM H-89, 100 nM wortmannin, or the combination thereof) for 15 min at 37°C followed by treatment with either vehicle (0.1% Me2SO) or 1 µM PGE2 for 10 min. The cells were washed with ice-cold phosphate-buffered saline and were placed on ice. Two hundred microliters of lysis buffer (20 mM MOPS, 50 mM 
-glycerolphosphate, 50 mM sodium fluoride, 1 mM sodium vanadate, 5 mM EGTA, 2 mM EDTA, 1% Nonidet P-40, 1 mM dithiothreitol, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) was added; after a 10-min incubation on ice, the cells were scraped off and transferred to microcentrifuge tubes. The cell lysates were centrifuged for 15 min at 16,000g, and aliquots of the supernatants containing 
0.2 µg of protein were assayed for PKA activity according to the manufacturer's instructions using an enzyme-linked immunosorbent assay kit and a synthetic peptide substrate for PKA (Stressgen Biotechnologies, San Diego, CA).
cAMP Assay. Cells were cultured in 12-well plates and were replaced with fresh Opti-MEM containing 0.1 mg/ml 3-isobutyl-1-methylxanthine (Sigma-Aldrich, St. Louis, MO). Cells were pretreated with either vehicle (0.1% Me2SO) or inhibitors (10 µM H-89, 100 nM wortmannin, or the combination thereof) for 15 min at 37°C followed by treatment with either vehicle (0.1% Me2SO) or 1 µM PGE2 for 10 min. The media were removed and the cells were placed on ice. Two hundred microliters of TE buffer (50 mM Tris-HCl and 4 mM EDTA, pH 7.5) was added and the cells were scraped off and transferred to microcentrifuge tubes. The samples were boiled for 8 min, placed on ice, and centrifuged for 1 min at 16,000g. Two microliters of the supernatants (representing 104 cells) were transferred to microcentrifuge tubes containing 48 µl of TE, 50 µl of [3H]cAMP (Invitrogen) and 100 µl of 0.06 mg/ml PKA (Sigma-Aldrich). The samples were vortexed, incubated on ice for 2 h, followed by the addition of 100 µl of TE buffer containing 2% bovine serum albumin and 26 mg/ml powdered charcoal (Sigma-Aldrich). After vortexing and centrifugation for 1 min at 16,000g, the radioactivity in 200-µl aliquots of the supernatants was determined by liquid scintillation spectrometry. The amount of cAMP present was calculated from a standard curve prepared using unlabeled cAMP and was expressed as picomoles per 104 cells.
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Results |
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; Johannessen et al., 2004). We have previously characterized the stimulation of intracellular cAMP formation by PGE2 in HEK cells stably expressing either the human EP2 or EP4 prostanoid receptor (Fujino et al., 2002). Although the levels of receptor expression were similar (100 fmol/mg of whole-cell protein), the maximal extent of stimulation of cAMP formation in the EP4-expressing cell line was only 20% of that obtained in the EP2-expressing cell line. However, for a separate measure of signaling activity (PGE2 stimulation of Tcf-mediated transcriptional activation) both cell lines yielded similar maximal extents of activation (Fujino et al., 2002). We were interested, therefore, in possible similarities or differences in the ability of PGE2 to stimulate the phosphorylation of CREB at Ser-133 in these same EP2- or EP4-expressing cell lines. For these experiments, cells expressing either the human EP2 or EP4 prostanoid receptor or untransfected HEK cells were treated with 1 µM PGE2 for various times ranging from 5 to 60 min and were then immunoblotted with antibodies that recognized either phospho-CREB (pCREB/pATF1) or total CREB (phosphorylated and nonphosphorylated). As shown in Fig. 1, A and B, treatment with PGE2 resulted in a rapid time-dependent phosphorylation of CREB in both the EP2 and EP4 receptor expressing cell lines. The time course of CREB phosphorylation was similar for both cell lines and seemed to reach a similar maximum. As shown in Fig. 1C, there was no significant effect of PGE2 on the phosphorylation of CREB in the untransfected HEK cells. Figure 1 also shows that when these blots were stripped and re-probed with antibodies that recognized both the phosphorylated and nonphosphorylated forms of CREB, nearly identical amounts of CREB were present throughout the time course of treatment and among the three cell lines.
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). In EP2-expressing cells, however, these activities are mediated primarily by a PKA-dependent pathway, whereas in EP4-expressing cells, these activities primarily involve the activation of a PI3K-dependent pathway. Therefore, we decided to use H-89, an inhibitor of PKA, and wortmannin, an inhibitor of PI3K, to investigate the contributions of PKA signaling and PI3K signaling, respectively, to the PGE2-stimulated phosphorylation of CREB in these cells. For these experiments, the cells were pretreated with either vehicle, 10 µM H-89, or 100 nM wortmannin for 15 min followed by treatment with 1 µM PGE2 for 10 min and were then immunoblotted with antibodies to phospho-CREB (pCREB/pATF1) or total CREB. As depicted in Fig. 3A, after the pretreatment of EP2-expressing cells with H-89 there was a significant decrease of PGE2-stimulated phosphorylation of CREB, suggesting the involvement of PKA in this phosphorylation. On the other hand, pretreatment of EP4-expressing cells with H-89 had no significant effect on PGE2-mediated CREB phosphorylation. Figure 3B shows that, somewhat unexpectedly, pretreatment with wortmannin had no effect on PGE2-stimulated CREB phosphorylation in either EP2- or EP4-expressing cells. We have shown in prior studies that pretreatment of EP4-expressing cells with 100 nM wortmannin for 15 min can inhibit the PGE2-mediated phosphorylation of GSK-3, as well as the PGE2-mediated stimulation of Tcf reporter gene activity and induction of EGR-1 (Fujino et al., 2002, 2003). As an additional precaution, however, we repeated the experiments depicted in Fig. 3B with the PI3K inhibitor LY294002. As with wortmannin, pretreatment of EP2- and EP4-expressing cells with 30 µM LY294002 for 15 min had no effect on the PGE2-stimulated phosphorylation of CREB (data not shown).
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Effects of H-89, Wortmannin and the Combination of H-89 and Wortmannin on PGE2 Stimulated PKA Activity in EP2 or EP4 Transfected HEK Cells. To further explore the potential interaction of the PKA and PI3K signaling pathways, we examined the effects of inhibitors of these pathways, both alone and in combination, on PGE2-stimulated PKA activity in cells stably expressing either the human EP2 or EP4 prostanoid receptor. As noted previously in Fig. 2, Fig. 5 shows that in the absence of any inhibitor pretreatment, there was a 2.6-fold stimulation of PKA activity in EP2 cells treated for 10 min with 1 µM PGE2 and a 1.5-fold stimulation of PKA activity in EP4-expressing cells. Pretreatment of both EP2- and EP4-expressing cells with H-89 alone resulted in a nearly complete inhibition of PGE2-stimulated PKA activity in both cell lines. The inhibition of PGE2-stimulated PKA activity in EP2-expressing cells correlated nicely with the inhibition of PGE2-stimulated CREB phosphorylation by H-89 in the EP2-expressing cells (compare Fig. 3A). On the other hand, the inhibition of PGE2-stimulated PKA activity in EP4-expressing cells did not correlate with the effects of H-89 on PGE2-stimulated CREB phosphorylation in EP4-expressing cells, which was not inhibited by H-89 pretreatment (compare Fig. 3A). These findings further support the conclusion that the PGE2-mediated phosphorylation of CREB in EP4-expressing cells does not occur through a PKA-dependent pathway.
As shown in Fig. 5, pretreatment with wortmannin had essentially no effect on PGE2-stimulated PKA activity in EP2-expressing cells, but in EP4-expressing cells, it caused a significant increase in PGE2-stimulated PKA activity compared with untreated cells. Thus, after pretreatment of EP4-expressing cells with wortmannin, PGE2-stimulated PKA activity increased approximately 2.7-fold, whereas in untreated cells, the stimulation was approximately 1.5-fold. These data may explain the failure of wortmannin pretreatment to block PGE2-stimulated CREB phosphorylation in EP4-expressing cells (Fig. 3B) because the PGE2-mediated activation of PI3K signaling in EP4-expressing cells inhibits the activity of PKA. Therefore, pretreatment of EP4-expressing cells with wortmannin alone relieves this PI3K-mediated inhibition, resulting in a PKA-dependent phosphorylation of CREB. As expected, Fig. 5 shows that pretreatment of EP2- and EP4-expressing cells with the combination of H-89 and wortmannin inhibited the PGE2 stimulation of PKA activity in both cell lines. These findings are consistent with the inhibition of PGE2-mediated CREB phosphorylation in both the EP2 and EP4 expressing cell lines after pretreatment with the combination of H-89 and wortmannin (compare Figure 4).
Effects of H-89, Wortmannin, and the Combination Thereof on PGE2-Stimulated cAMP Formation in EP2- or EP4-Transfected HEK Cells. Given that the activity of PKA is regulated by cAMP, we decided to examine PGE2-stimulated cAMP formation in EP2 and EP4 cells under control conditions and after treatment with inhibitors of the PKA and PI3K pathways. This was of particular interest with respect to wortmannin's effect of increasing PGE2-stimulated PKA activity in EP4-expressing cells, because this could reflect either a PI3K-mediated inhibition of PKA activity or a PI3K-mediated increase in intracellular cAMP accumulation. As shown previously in Fig. 2, Fig. 6 shows that in the absence of pretreatment with inhibitors, there was a 23-fold stimulation of cAMP accumulation in EP2 cells treated for 10 min with 1 µM PGE2 and a 7.4-fold stimulation of cAMP accumulation in EP4-expressing cells. For both EP2- and EP4-expressing cells, the PGE2-mediated stimulation of cAMP accumulation correlated reasonably well with the PGE2-mediated stimulation of PKA activity shown in Fig. 5. Figure 6 also shows that pretreatment with either H-89, wortmannin, or both had virtually no effect on PGE2-stimulated cAMP accumulation in either EP2- or EP4-expressing cells. The evidence that wortmannin pretreatment of EP4-expressing cells had little effect on PGE2-mediated cAMP accumulation supports the conclusion that the PGE2-mediated activation of PI3K signaling in EP4-expressing cells inhibits the activity of PKA by a mechanism that does not involve a decreased formation of intracellular cAMP.
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Discussion |
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s-coupled GPCRs is, in evolutionary terms, perhaps the oldest and most wide-spread second-messenger pathway used by this superfamily of receptors. For example, of the eight most closely related human prostanoid receptor subtypes, four of them (EP2, EP4, IP, and DP1) couple primarily to this signaling pathway (Hata and Breyer, 2004). Three of the remaining prostanoid receptor subtypes (EP1, FP, and TP) couple primarily to Gq and activate the inositol phosphate signaling pathway; one (EP3) couples to Gi and decreases the formation of intracellular cAMP through the inhibition of adenylyl cyclase. It is interesting that the phylogeny of these prostanoid receptors shows that the subtypes that couple to Gs are all more closely related to each other and form a distinct subfamily compared with the subtypes that couple to Gq and Gi (Regan et al., 1994; Toh et al., 1995). The fact that EP receptor subtypes are present in both major subfamilies suggests that the primordial receptor was an EP subtype and that the initial evolution of these receptors was based on their ability to activate different signal transduction pathways (Regan et al., 1994).
Of the four prostanoid receptor subtypes that couple to Gs, two (EP2 and EP4) are activated by PGE2 and two (IP and DP1) are activated by prostacyclin and PGD2, respectively. As the discovery of these prostanoid receptor subtypes unfolded, it seemed likely that the IP and DP1 receptors evolved to subserve different signaling molecules, which could also be said of the EP2 and EP4 receptors as a group, but obviously not as individual subtypes. In fact, the EP2 and EP4 receptors do not seem to have evolved together as their own subfamily, because the phylogeny shows that the EP2, IP, and DP1 receptors are actually more related to each other and form a subgroup distinct from the EP4 receptor (Toh et al., 1995). This suggests an evolutionary divergence of an ancestral Gs-coupled EP receptor into two descendants. One of these descendants was a Gs-coupled EP receptor that eventually gave rise to the EP2, IP, and DP1 receptor subtypes; the second descendant was a Gs-coupled EP receptor that evolved into the present day EP4 receptor subtype. Thus, the evolution of the EP2, IP, and DP1 receptor subtypes, presumably on the basis of their ability to discriminate between their respective endogenous ligands, seems to have come after an earlier event that eventually gave rise to the EP2 and EP4 subtypes. The underlying basis for the initial divergence of the ancestral Gs coupled EP receptor is speculative, but it is reasonable to suppose that it may have been a consequence of functional differences involving receptor regulation or signal transduction.
As reviewed in the Introduction, the initial characterization of the EP2 and EP4 receptors subtypes revealed no significant functional differences between these receptors; they were both preferentially activated by PGE2 and they both stimulated the formation of intracellular cAMP (Honda et al., 1993; Regan et al., 1994). Thereafter, it was found that there were differences in the desensitization (Nishigaki et al., 1996) and internalization (Desai et al., 2000) of these receptors; more recently, it has become clear that there are significant differences in the signaling properties of the EP2 and EP4 prostanoid receptors (Fujino and Regan, 2003). As shown in Fig. 7, what seems to be emerging with respect to the signaling differences is that the EP2 receptor subtype couples to a classic cAMP signaling pathway involving a marked stimulation of intracellular cAMP formation and activation of PKA. The EP4 receptor, on the other hand, can activate the cAMP/PKA pathway but it is less robust, and there is a concomitant activation of the PI3K and ERK signaling pathways. The stimulation of either receptor subtype often leads to the activation of the same downstream effectors, albeit by different pathways. For example, both the EP2 and EP4 receptors can stimulate Tcf transcriptional activation, but the EP2 receptor uses primarily a cAMP/PKA-dependent pathway, whereas the EP4 receptor uses primarily a PI3K-dependent pathway (Fujino et al., 2002). However, stimulation of the EP4 receptor can also result in the selective activation of downstream effectors that are not activated after the stimulation of EP2 receptors. For example, PGE2 stimulation of the EP4 receptor induces the expression of EGR-1, which does not occur after PGE2 stimulation of the EP2 receptor (Fujino et al., 2003).
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2-adrenergic receptor in rat cardiomyocytes, which is also a Gs coupled receptor that can activate cAMP/PKA signaling (Jo et al., 2002). Thus, inhibition of PI3K with wortmannin resulted in a marked increase in the PKA-mediated phosphorylation of phospholamban after agonist stimulation of the 2-adrenergic receptor. The apparent increase in PKA activity occurred in the absence of any change in intracellular cAMP formation and suggests that agonist stimulation of the 2-adrenergic receptor negatively regulates the activity of PKA through the activation of a PI3K signaling pathway.
The phosphorylation of CREB on Ser-133 is central to the regulation of CREB-mediated transcriptional activation and correlates well with the extent of target gene activation (Mayr and Montminy, 2001; Johannessen et al., 2004). The stimulation of intracellular cAMP formation and activation of PKA by Gs-coupled GPCRs is a key mediator of the Ser-133 phosphorylation of CREB; as reviewed in the introduction, however, it is not the only mechanism. This is clearly exemplified by our present findings, which show that the EP2 receptor-mediated phosphorylation of CREB on Ser-133 is mainly PKA-dependent, whereas the EP4-mediated CREB phosphorylation is not and involves a PI3K-dependent pathway. The activation of Ras signaling pathways by growth factor receptors has been shown to promote the phosphorylation of CREB on Ser-133, and we have reported previously the PI3K-dependent activation of ERKs 1 and 2 by the EP4 receptor but not the EP2 receptor (Fujino et al., 2003). However, inhibition of mitogen-activated protein kinase kinase signaling with PD98059, did not affect the PGE2-mediated phosphorylation of CREB in either the EP2- or EP4-expressing cells (data not shown). We found previously that the inhibition of MEK by PD98059 in EP4-expressing cells blocked the ERK-mediated induction of EGR-1 expression, which suggests that activation of a mitogen-activated protein kinase kinase/ERK signaling pathway is not involved in the PGE2-mediated phosphorylation of CREB in EP4-expressing cells.
It is well established that the serine/threonine kinase, protein kinase B (Akt), can phosphorylate CREB on Ser-133 in response to a variety of stressful stimuli (Mayr and Montminy, 2001; Johannessen et al., 2004). Akt itself is phosphorylated and activated by the phosphoinositide-dependent kinase-1 (PDK-1) as a downstream consequence of the activation of PI3K (Toker, 2000). We have shown previously that treatment of EP4-expressing cells with PGE2 stimulates the phosphorylation of Akt and that this phosphorylation can be blocked by pretreatment with wortmannin (Fujino et al., 2002). As shown in Fig. 7, it is plausible that the PGE2-stimulated phosphorylation of CREB in EP4-expressing cells is mediated by Akt after the activation of PI3K signaling. The specific PI3K and the mechanism of its activation by the EP4 receptor remains unknown. PI3K actually comprises a family of enzymes, most of which can be inhibited by wortmannin (Vanhaesebroeck and Waterfield, 1999). GPCRs have been shown to activate PI3K by several mechanisms including direct activation by G-protein 
-subunits and transactivation through the epidermal growth factor receptor. Further studies will be needed to elucidate the mechanism of PI3K activation after stimulation the EP4 receptor with PGE2.
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Footnotes |
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS: GPCR, G-protein-coupled receptor; PG, prostaglandin; GSK, glycogen synthase kinase; Tcf, T cell factor; PI3K, phosphatidylinositol 3-kinase; EGR, early growth response factor; ERK, extracellular signal-regulated kinase; CRE, cAMP response element; CREB, cAMP response element-binding protein; PKA, protein kinase A; HEK, human embryonic kidney; Me2SO, dimethyl sulfoxide; MOPS, 3-(N-morpholino)-propanesulfonic acid; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; PD98059, 2'-amino-3'-methoxyflavone.
Address correspondence to: Dr. John W. Regan, Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ 85721–0207. E-mail: regan{at}pharmacy.arizona.edu
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Desai S, April H, Nwaneshiudu C, and Ashby B (2000) Comparison of agonist-induced internalization of the human EP2 and EP4 prostaglandin receptors: role of the carboxyl terminus in EP4 receptor sequestration. Mol Pharmacol 58: 1279–1286.
Fujino H and Regan JW (2003) Prostanoid receptors and phosphatidylinositol 3-kinase: a pathway to cancer? Trends Pharmacol Sci 24: 335–340.[CrossRef][Medline]
Fujino H, West KA, and Regan JW (2002) Phosphorylation of glycogen synthase kinase-3 and stimulation of T-cell factor signaling following activation of EP2 and EP4 prostanoid receptors by prostaglandin E2. JBiol Chem 277: 2614–2619.
Fujino H, Xu W, and Regan JW (2003) Prostaglandin E2 induced functional expression of early growth response factor-1 by EP4, but not EP2, prostanoid receptors via the phosphatidylinositol 3-kinase and extracellular signal-regulated kinases. J Biol Chem 278: 12151–12156.
Hata AN and Breyer RM (2004) Pharmacology and signaling of prostaglandin receptors: multiple roles in inflammation and immune modulation. Pharmacol Ther 103: 147–166.[CrossRef][Medline]
Honda A, Sugimoto Y, Namba T, Watabe A, Irie A, Negishi M, Narumiya S, and Ichikawa A (1993) Cloning and expression of cDNA for mouse prostaglandin E receptor EP2 subtype. J Biol Chem 268: 7759–7762.
Jo SH, Leblais V, Wang PH, Crow MT, and Xiao RP (2002) Phosphatidylinositol 3-kinase functionally compartmentalizes the concurrent Gs signaling during 2-adrenergic stimulation. Circ Res 91: 46–53.
Johannessen M, Delghandi MP, and Moens U (2004) What turns CREB on? Cell Signal 16: 1211–1227.[CrossRef][Medline]
Mayr B and Montminy M (2001) Transcriptional regulation by the phosphorylation-dependent factor CREB. Nat Rev Mol Cell Biol 2: 599–609.[CrossRef][Medline]
Nishigaki N, Negishi M, and Ichikawa A (1996) Two Gs-coupled prostaglandin E receptor subtypes, EP2 and EP4, differ in desensitization and sensitivity to the metabolic inactivation of the agonist. Mol Pharmacol 50: 1031–1037.[Abstract]
Pozzi A, Yan X, Macias-Perez I, Wei S, Hata AN, Breyer RM, Morrow JD, and Capdevila JH (2004) Colon carcinoma cell growth is associated with prostaglandin E2/EP4 receptor-evoked ERK activation. J Biol Chem 279: 29797–29804.
Regan JW (2003) EP2 and EP4 prostanoid receptor signaling. Life Sci 74: 143–153.[CrossRef][Medline]
Regan JW, Bailey TJ, Pepperl DJ, Pierce KL, Bogardus AM, Donello JE, Fairbairn CE, Kedzie KM, Woodward DF, and Gil DW (1994) Cloning of a novel human prostaglandin receptor with characteristics of the pharmacologically defined EP2 subtype. Mol Pharmacol 46: 213–220.[Abstract]
Sheng H, Shao J, Washington MK, and DuBois RN (2001) Prostaglandin E2 increases growth and motility of colorectal carcinoma cells. J Biol Chem 276: 18075–18081.
Toh H, Ichikawa A, and Narumiya S (1995) Molecular evolution of receptors for eicosanoids. FEBS Lett 361: 17–21.[CrossRef][Medline]
Toker A (2000) Protein kinases as mediators of phosphoinostide 3-kinase signaling. Mol Pharmacol 57: 652–658.
Vanhaesebroeck B and Waterfield MD (1999) Signaling by distinct classes of phosphoinositide 3-kinases. Exp Cell Res 253: 239–254.[CrossRef][Medline]
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 K. M. Ansari, Y. M. Sung, G. He, and S. M. Fischer Prostaglandin receptor EP2 is responsible for cyclooxygenase-2 induction by prostaglandin E2 in mouse skin Carcinogenesis, October 1, 2007; 28(10): 2063 - 2068. [Abstract] [Full Text] [PDF]
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 S. Donnini, F. Finetti, R. Solito, E. Terzuoli, A. Sacchetti, L. Morbidelli, P. Patrignani, and M. Ziche EP2 prostanoid receptor promotes squamous cell carcinoma growth through epidermal growth factor receptor transactivation and iNOS and ERK1/2 pathways FASEB J, August 1, 2007; 21(10): 2418 - 2430. [Abstract] [Full Text] [PDF]
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 R. Rao, R. Redha, I. Macias-Perez, Y. Su, C. Hao, R. Zent, M. D. Breyer, and A. Pozzi Prostaglandin E2-EP4 Receptor Promotes Endothelial Cell Migration via ERK Activation and Angiogenesis in Vivo J. Biol. Chem., June 8, 2007; 282(23): 16959 - 16968. [Abstract] [Full Text] [PDF]
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 Z Cheng, E L Sheldrick, E Marshall, D C Wathes, D R E Abayasekara, and A P F Flint Control of cyclic AMP concentration in bovine endometrial stromal cells by arachidonic acid Reproduction, May 1, 2007; 133(5): 1017 - 1026. [Abstract] [Full Text] [PDF]
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 S. Huang, S. H. Wettlaufer, C. Hogaboam, D. M. Aronoff, and M. Peters-Golden Prostaglandin E2 inhibits collagen expression and proliferation in patient-derived normal lung fibroblasts via E prostanoid 2 receptor and cAMP signaling Am J Physiol Lung Cell Mol Physiol, February 1, 2007; 292(2): L405 - L413. [Abstract] [Full Text] [PDF]
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Y. Murakami, H. Kohsaka, H. Kitasato, and T. Akahoshi Lipopolysaccharide-Induced Up-Regulation of Triggering Receptor Expressed on Myeloid Cells-1 Expression on Macrophages Is Regulated by Endogenous Prostaglandin E2 J. Immunol., January 15, 2007; 178(2): 1144 - 1150. [Abstract] [Full Text] [PDF]
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 L. Yang, Y. Huang, R. Porta, K. Yanagisawa, A. Gonzalez, E. Segi, D. H. Johnson, S. Narumiya, and D. P. Carbone Host and Direct Antitumor Effects and Profound Reduction in Tumor Metastasis with Selective EP4 Receptor Antagonism Cancer Res., October 1, 2006; 66(19): 9665 - 9672. [Abstract] [Full Text] [PDF]
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 J.-Y. Qian, A. Leung, P. Harding, and M. C. LaPointe PGE2 stimulates human brain natriuretic peptide expression via EP4 and p42/44 MAPK Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H1740 - H1746. [Abstract] [Full Text] [PDF]
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 H. Fujino and J. W. Regan EP4 Prostanoid Receptor Coupling to a Pertussis Toxin-Sensitive Inhibitory G Protein Mol. Pharmacol., January 1, 2006; 69(1): 5 - 10. [Abstract] [Full Text] [PDF]
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, p < 0.05, t test.
, p < 0.05, t test.
